CYP2C19-rs4986893 confers risk to major depressive disorder and bipolar disorder in the Han Chinese population whereas ABCB1-rs1045642 acts as a protective factor

Background Genetic risks may predispose individuals to major mood disorders differently. This study investigated the gene polymorphisms of previously reported candidate genes for major depressive disorder (MDD) and bipolar disorder (BPD) in the Han Chinese population. Methods Twenty loci of 13 candidate genes were detected by MALDI-TOF mass spectrometry in 439 patients with MDD, 600 patients with BPD, and 464 healthy controls. The distribution of genotypes in alleles, Hardy-Weinberg equilibrium, and genetic association were analyzed using the PLINK software. The linkage of disequilibrium and haplotype analyses were performed using the Haploview software. Results Out of the 20 loci analyzed, CYP2C19-rs4986893, ABCB1-rs1045642, and SCN2A-rs17183814 passed Bonferroni correction; their statistical powers were > 55%. The minor allele frequencies (MAF) of CYP2C19-rs4986893 in the MDD group (0.0547) and BPD group (0.0533) were higher than that of the control group (0.0259, P < 0.05), leading to the odds ratios (ORs) of MDD (2.178) and BPD (2.122), respectively. In contrast, the lower MAFs of ABCB1-rs1045642 were observed in both MDD (0.3599, OR = 0.726) and BPD (0.3700, OR = 0.758) groups than controls (0.4364, P < 0.05). The MDD group had a higher MAF of SCN2A-rs17183814 than controls (0.1743 vs. 0.1207, OR = 1.538, P < 0.05). Moreover, a G-A haplotype composed by CYP2C19-rs4986893 and -rs4244285 was associated with BPD (OR = 1.361, P < 0.01), and the A-G haplotype increased the risks to both MDD (OR = 2.306, P < 0.01) and BPD (OR = 2.332, P < 0.001). The CYP2C19 intermediate metabolizer and poor metabolizer (IM&PM) status was related to the raised risk of both MDD (OR = 1.547, P < 0.01) and BPD (OR = 1.808, P < 0.001). Conclusion Our data indicate that the impaired CYP2C19 metabolism caused by the haplotypes integrated by CYP2C19 alleles might confer the risk to MDD and BPD, whereas the ABCB1-rs1045642 T allele serves as a protective factor.

Keywords Major depressive disorder, Bipolar disorder, Genetics, Risk factor, Susceptibility

Background
Major depressive disorder (MDD) and bipolar disorder (BPD) are chronic and recurrent mood disorders affecting approximately 13% of the world's population [1,2]. Due to the substantial increase in healthcare expenditure and increasing suicide rate from MDD and BPD, these mood disorders form a tremendous socioeconomic burden on families and society. MDD is characterized by significant and persistent depressed mood, waned interest, slowed thinking, and cognitive impairment [3], whereas BPD is characterized by extreme mood state swing between mania and depression [4]. Previous studies have suggested that these two mood disorders do not only have overlapping symptoms but also share mechanisms [5], including metabolic dysregulation, insulin resistance, immune disorders, and neural signal transduction pathway malfunction. Several genes closely related to pathological mechanisms have been identified in previous studies. For example, polymorphisms of CYP2C19, CYP2C9, NAT2, UGT1A9, and ABCB1 related to the activation or detoxification of drugs and endogenous substances have emerged as major genetic factors in several psychiatric disorders [6][7][8].
Since genetic factors with accumulative multiple variants clearly play a critical role in the etiology and pathology of polygenic mood disorders [9], characterization of the genetic features involved in etiological mechanism is particularly required. However, the potential genetic associations remain unclear, and the results of genomewide association studies (GWAS) on mood disorders are rarely repeatable [9]. Furthermore, several studies failed to identify gene-disease correlations in patients with mood disorders [10,11]. One probable reason for the unsuccessful generation of repeatable results to demonstrate the main effects of these genes on these diseases is that allelic frequencies may vary in different racial and ethnic backgrounds. The results from previous studies on particular genetic backgrounds cannot be applied to other populations. In this study, we investigated the candidate genes in the Han Chinese population. An effective method was developed to simultaneously analyze the pathogenic effect of these specific genes for constructing a custom single nucleotide polymorphism (SNP) detection package covering loci selected based on current assumptions and proofs from previous studies. Twenty loci from LEPR, SCN2A, SCN1A, UGT1A9, GSK3B, HLA-B, ABCB1, NAT2, CYP2C19, CYP2C9, ANKK1, SH2B1, and INSR were present in the SNP detection array.
In terms of the study on genetic risk could be pathogenesis support and diagnostic reference for psychiatric diseases, this study verifies the association between SNPs in 13 candidate genes and the risk of mood disorders, including MDD and BPD, in the Han Chinese population via MALDI-TOF mass spectrometry. Additionally, the effect of haplotypes and metabolism statuses were analyzed.

Study participants
All participants were Han Chinese living in Guangdong Province, Southern China. The case group included 439 patients with MDD (158 males and 281 females) and 600 patients with BPD (258 males and 342 females) hospitalized at the Affiliated Brain Hospital of Guangzhou Medical University from February 2020 to September 2021. The diagnosis for each patient was strictly based on the DSM-V criteria [12,13] for MDD and BPD, and was agreed by at least two independent and experienced psychiatrists. Patients were excluded if they were diagnosed with primary or comorbid physical diseases or other mental illnesses, such as schizoaffective disorder, schizophrenia, dementia, alcohol or drug addiction, posttraumatic stress disorder, obsessive-compulsive disorder, panic disorder, and anxiety disorder. The control group consisted of 464 adults (196 males and 268 females) who underwent annual physical examinations, and those with personal or family history of major psychiatric disorders were excluded. The corresponding mean ages of the control, MDD, and BPD groups were 30.7 ± 12.6 years, 29.8 ± 14.9 years, and 30.5 ± 14.7 years, respectively. Age and gender were matched between the case and control groups (P > 0.05). Demographic and clinical data of MDD and BPD cases are listed in Table 1.

DNA extraction and SNP genotyping
EDTA-K 2 anticoagulant blood, 2 mL, was collected from all participants for SNP detection. DNA was extracted from 0.5 mL of blood using the Blood Genomic DNA Isolation Kit (Shanghai BaiO Technology Co. Ltd), following the manufacturer's manual. The samples were kept at − 80℃ until further analysis.
DNA samples were diluted to 5 ng/uL and then used for amplification. After the multiplex PCRs were performed, the products were treated with shrimp alkaline phosphatase to remove excess dNTPs and used as templates for the primer extension reactions using iPLEX mixture. The final products were automatically spotted on the MassARRAY SpectroCHIP. The target panels were inserted into the MALDI-TOF mass spectrometer, and SNP data were auto-analyzed by this instrument. Shanghai Kangli Medical Research Institute assisted with SNP genotyping. Twenty loci from LEPR, SCN2A, SCN1A, UGT1A9, GSK3B, HLA-B, ABCB1, NAT2, CYP2C19, CYP2C9, ANKK1, SH2B1, and INSR genes were typed.

Statistical analysis
Age difference was compared using the student's t-test, and gender and haplotype were analyzed with Pearson's Chi-square test using the IBM SPSS (IBM, Armonk, NY) version 20. Hardy-Weinberg equilibrium analysis, genotype and allele frequencies, and association tests were conducted using the PLINK software version 1.9 (https:// www. cog-genom ics. org/ plink) [30]. Exact test was used for Hardy-Weinberg equilibrium analysis in PLINK software. The linkage disequilibrium and haplotype analysis were performed using the Haploview software (Broad, Cambridge, MA) version 4.2 [31]. The P values of alleles were corrected by Bonferroni correction, in which the adjusted P values acquired were multiplied by SNP amount. Statistical power was calculated using the PS program on line (https:// statc omp2. app. vumc. org/ ps/).

Hardy-Weinberg equilibrium analysis of 20 SNPs in all groups
Hardy-Weinberg equilibrium of 20 SNPs was tested ( Table 2). The SNPs passed the Hardy-Weinberg equilibrium test in all groups, showed that sample sets were representative of the population. The GRCh38 human reference genome was used for genetic variant location. ID number and position of SNPs are shown in Table 2.

Association analysis of genetic predisposition in MDD and BPD
The genotype distribution and minor allele frequencies (MAF) of each SNP are listed in Table 3. After Bonferroni correction, only CYP2C19-rs4986893, ABCB1-rs1045642, and SCN2A-rs17183814 were passed for subsequent analysis; their statistical powers were greater than 55%. The MAF of CYP2C19-rs4986893 in the MDD group (0.0547) and BPD group (0.0533) were higher than that of the control group (0.0259, P < 0.05).
With the control group as reference, participants with the CYP2C19-rs4986893 A allele had odds ratios (ORs) of 2.178 and 2.122 for MDD and BPD, respectively. In contrast, both MDD (0.3599) and BPD (0.3700) groups had lower MAFs of ABCB1-rs1045642 than the control (0.4364, P < 0.05) group. Therefore, participants with the ABCB1-rs1045642 T allele had ORs of 0.726 and 0.758 for MDD and BPD, respectively. The MAF of the SCN2A-rs17183814 in patients with MDD (0.1743) was higher than that of the controls (0.1207, P < 0.05). Participants with the SCN2A-rs17183814 A allele had a 1.538-fold greater risk to suffer from MDD than those without it.

CYP2C19 metabolizer status distribution in controls and cases
When genotypes composed of rs4244285 and rs4986893 were translated into predicted CYP2C19 metabolism, it could be categorized as normal metabolizer (NM), intermediate metabolizer (IM), poor metabolizer (PM). NM was the subject carried none of these defective alleles, while IM was the subject had one defective allele and PM was the one had two defective alleles. The distributions of these CYP2C19 metabolizer statuses were significantly different between cases and controls. The frequencies of IM&PM status were higher in MDD (57.40%, OR = 1.547) and BPD (61.17%, OR = 1.808) cases than those in controls (46.55%, P < 0.05), showed in Table 5.

Discussion
Many etiopathogenetic mechanisms are involved in mood disorders, such as MDD and BPD. Due to the common symptoms and shared etiologies between these disorders, we sought to clarify whether correlations existed between candidate genetic variants in selected genes and susceptibility to MDD and BPD in the Han Chinese population. To the best of our knowledge, this study is the first Chinese study to examine the implication of 13 genes on both MDD and BPD risk, covering LEPR, SCN2A, SCN1A, UGT1A9, GSK3B, HLA-B, ABCB1, NAT2, CYP2C19, CYP2C9, ANKK1, SH2B1, and INSR. Our data suggested that SCN2A-rs17183814, ABCB1-rs1045642, χ 2 , P, OR, and 95%CI in this table indicate chi-square value, P value, odds ratio, and 95% confidence interval of minor alleles, respectively MAF Minor allele frequency  and CYP2C19-rs4986893 had associations with MDD or BPD, providing evidence for genetic vulnerability to mood disorders, and provided a basis for understanding the etiology of these disorders for earlier prevention. The neuronal voltage-gated sodium channel, which modulates neuron excitability and initial transduction, is encoded by the SCN2A gene expressed in the initial segment of the axon and plays a crucial part in neuronal pathfinding and neurite outgrowth [32]. Once neuronal voltage-gated sodium channels are deficient in mature neurons, action potential is back-propagated, dendrite excitability is reduced, and synaptic efficacy is damaged [33]. Diminished channel function interferes with the neural signal transduction pathway, resulting in the occurrence of MDD, BPD, and autism spectrum disorder [26,34]. Our data could not confirm the association between SCN2A-rs17183814 and BPD in European and Chinese Han populations [26]. We thought the reasons for the discrepancy might be due to the heterogeneity in the cases, the differences in racial composition and the smaller sample sizes than Zhao's study (1146 BPD cases and 1956 controls). Additionally, we found that the A allele of SCN2A-rs17183814 increased the odds of developing MDD by 1.583-fold, which was different from the contribution of the G allele to the prevalence of MDD (OR = 1.116) observed by Zhao [26]. The biological link between the locus and affective disorders needs further clarification.
ABCB1, which encodes a permeability glycoprotein that is highly expressed in the brain for exporting various hydrophobic compounds, plays a vital role in forming a protective physiological barrier and emerges as an active eliminator for xenobiotics and cellular metabolites [35]. The vulnerability to MDD can be predicted with ABCB1 by altering the activity of the hypothalamic-pituitaryadrenal axis [36]. The C allele of the ABCB1-rs1045642 polymorphism was connected with boosted interpersonal sensitivity among Japanese populations [8]; this allele has been generally accepted as one of the vulnerability factors for depression. Ozbey et al. [37] showed in a Turkish population that ABCB1-rs1045642 C allele and CC genotype were associated with susceptibility to the development of MDD. In addition, a study using a mouse model has shown that higher cortisol levels accumulated in the plasma and brain of ABCB1 -/-knockout mice [18]. Based on these findings, we assumed that the ABCB1-rs1045642 C allele over-expresses the permeability glycoprotein, restricting the entry of cortisol into the brain. This leads to a lower cortisol level in the brain and higher interpersonal sensitivity. Negative feedback from the lower cortisol level can lead to a hyperactive hypothalamic-pituitary-adrenal axis, which promotes the release   [38]. A Chinese study showed that the TG haplotype of rs1045642-rs2032582 carriers reduced MDD risk by approximately 53% [39]. Our results were consistent with those of previous studies, where the T allele lowered the risks for MDD and BPD by 0.726-and 0.758-fold, respectively. However, the association between ABCB1-rs1045642 polymorphism and mood disorders has not been established. Some studies have suggested that ABCB1-rs1045642 T allele as the variant contributes to the predisposition of MDD [40,41]. The CYP2C19 enzyme plays a critical role in metabolizing not only drugs or xenobiotics that affect therapeutic outcomes but also endogenous substrates containing steroid hormones, vitamin D, eicosanoids, arachidonic acids, and cholesterol that could also confer susceptibility to many diseases [42,43]. Recent studies have suggested that impaired CYP2C19 metabolizers had higher self-rated Beck Depression Inventory-II scores than normal metabolizers. Damaged CYP2C19 enzyme activity was associated with more severe MDD, despite CYP2C19-metabolized medication treatment and treatment discrepancy status [7]. CYP2C19 polymorphism has been demonstrated to affect the conversion and degradation of endogenous compounds, including psychoactive steroid hormones (e.g. estrone, estradiol, progesterone, and testosterone) in in vitro studies [44]. Our study findings suggest that the A allele of CYP2C19-rs4986893 had a 2.178-fold higher prevalence of MDD and 2.122-fold increased possibility of BPD occurrence. The haplotypes of rs4986893-G and rs4244285-A might increase the risk for BPD, while the rs4986893-A and rs4244285-G haplotype soared both the risks of MDD and BPD. Additionally, the frequencies of IM&PM status were higher in MDD and BPD cases than those in controls, which also meant defective allele (rs4986893 or rs4244285) was related to the raised risk of both MDD and BPD. Our hypothesis is that the A allele of CYP2C19-rs4986893, as a variant, encodes impaired CYP2C19 enzyme, promoting steroid hormone disequilibrium, and resulting in a change in hypothalamic-pituitary-adrenal axis activity and mood disorder development. The hypothesis could be verified by the G-A and A-G haplotype found in the current study, which carried genetic variant and induced impaired metabolic enzyme activity. Nevertheless, contrasting results indicated that elevated CYP2C19 expression is related to depressive symptoms [45,46]. These deviations can be explained by inter-study discrepancies in CYP2C19 frequency or study methods. This study had several limitations. First, the channel function caused by SCN2A-rs17183814 mutation was not examined, and the substrate concentrations due to ABCB1-rs1045642 and CYP2C19-rs4986893 polymorphisms were not measured. These would have helped to characterize the physiological mechanisms. Second, the location of 20 candidate loci in various chromosomes make analysis of the effect of haplotypes on diseases difficult. Third, this study did not include controversial risk genes for depression, such as SLC6A4 and 5-HTTLPR; therefore, further investigations are needed [10,14].
In conclusion, we have provided additional evidence for genetic association, confirming that the CYP2C19-rs4986893 A allele is a risk factor and ABCB1-rs1045642 T allele is protective for MDD. For the first time, we showed that these two variants have a similar effect on BPD. Additionally, the SCN2A-rs17183814 A allele was found to increase the morbidity of MDD. The haplotype integrated by CYP2C19 alleles, and the CYP2C19 metabolizer status which was categorized as IM or PM might contribute to the risk of developing mood disorder.